Zucker Fatty Rats Research Articles

Abstract 17741: Angiotensin II Enables Leukocyte/Endothelium Integrated Signaling Mechanisms That are Causative of Endothelial Dysfunction in Insulin Resistance

Recent studies have linked vascular AngII/AT1r signaling to the calcium-dependent protease µ-calpain. We have recently reported that activation of endothelial-expressed µ-calpain by AngII/AT1r signaling induces leukocyte-endothelium interactions in the post-capillary venules of the microcirculation. Accordingly, we hypothesized that: 1) AngII/calpain signaling impairs endothelium-dependent relaxation in the arterial system of the obese, insulin resistant Zucker Fatty (ZF) rat, and 2) neutrophil derived myeloperoxidase (MPO) synergizes with AngII/AT1r signaling to sustain calpain activation in the vascular wall of ZF rats. We used wire myography to study endothelium-dependent relaxation and vascular reactivity of the rat and mouse aorta. Aortic rings isolated from ZF rats exhibited reduced endothelium-dependent relaxation to ACh and increased contractility to Norepinephrine (p<0.01 versus control Lean Zucker (ZL) rats). Incubation of aortic rings with either 1-µm AT1r blocker losartan or 10-µM calpain inhibitor ZLLal restored endothelium-dependent relaxation in aortic rings from ZF rats (p<0.05 versus untreated ZF aortic rings). Furthermore, endothelium-dependent relaxation to ACh was preserved in µ-calpain deficient mice fed a high-fat diet for 12 consecutive weeks. Plasma and aortic levels of MPO were also elevated in ZF rats (p<0.01 versus ZL rats) and reduced by losartan treatment (p<0.05 versus untreated ZF rat). Ex vivo studies demonstrated that AngII acutely induces MPO release from control neutrophils, a phenomenon that was prevented by pre-incubation of neutrophils with 1-µM Losartan. Biochemical studies in rat aortic endothelial cells confirmed that MPO significantly synergizes AngII/AT1r-induced calpain activation. Taken together our data demonstrate that AngII/AT1r signaling impairs endothelium-dependent relaxation in the obese, insulin resistant organism by a mechanism that implicates neutrophil-derived MPO and endothelial expressed calpain. These data uncover: 1) a novel integrative mechanisms responsible for hypertension in obesity with insulin resistance and 2) new molecular targets of the pleiotropic actions of AT1r blocker on the blood-vascular system.

Abstract 18067: Targeting Mitochondrial DNA to Reduce Consequences of Oxidative Stress: Role in the Prevention of the Diabetic Cardiomyopathy

Mitochondrial dysfunction and increased oxidative stress are hallmarks of diabetic cardiomyopathy, a condition characterized by increased risk of heart failure, independent of coronary atherosclerosis. Mitochondria not only are the major site of energy production, but also generate reactive oxygen species (ROS), and metabolic signals that produce coronary metabolic vasodilation (coupling of flow to metabolism). Accordingly, we hypothesized that elevations in mitochondrial ROS in obesity and diabetes produce oxidative mitochondrial DNA (mtDNA) injury (fragmentation); thus by repairing mtDNA fragmentation, we would restore coronary metabolic dilation. To test this hypothesis, we studied Zucker fatty obese rats (ZOF), lean littermates (ZL), and ZOF in which mtDNA fragmentation was repaired by administration of a cell permeable recombinant protein containing endonucleaseIII and a mitochondrial localization sequence (mt-tat-ENDOIII), which repairs fragmented mtDNA and restores mitochondrial functions. ZOF were treated daily for 3 days (1 µg/g body weight of mt-tat-ENDOIII). ZOF and ZL rats treated with vehicle were positive and negative controls, respectively. The product of mean arterial pressure and heart rate was used as a surrogate of cardiac work (CW). Myocardial blood flow (MBF) was measured by contrast echocardiography under baseline conditions (hexamethonium) and during NE infusion (i.v. norepinephrine, NE) to increase CW. During metabolic stress, ZOF showed coronary insufficiency compared to ZL (P<0.05). Importantly, coronary metabolic dilation in ZOF was restored by the administration of mt-tat-ENDOIII versus ZL (P=NS). We conclude that mitochondrial dysfunction participates to decrease the efficiency of the coronary metabolic regulation in diabetes. By preventing mt-DNA damage, adequate coronary metabolic dilatation can be restored; this could be a therapeutic approach for the prevention of diabetic cardiomyopathy.

Effect of Rosiglitazone on Bone Quality in a Rat Model of Insulin Resistance and Osteoporosis

OBJECTIVERosiglitazone (RSG) is an insulin-sensitizing drug used to treat type 2 diabetes mellitus. The A Diabetes Outcome Progression Trial (ADOPT) shows that women taking RSG experienced more fractures than patients taking other type 2 diabetes drugs. These were not osteoporotic vertebral fractures but, rather, occurred in the limbs. The purpose of this study was to investigate how RSG treatment alters bone quality, which leads to fracture risk, using the Zucker fatty rat as a model.RESEARCH DESIGN AND METHODSA total of 61 female 4-month-old rats were divided into six groups. One Sham group was a control and another was administered oral RSG 10 mg/kg/day. Four ovariectomized (OVX) groups were dosed as follows: controls, RSG 10 mg/kg, alendronate (ALN, injected at 0.7 mg/kg/week), and RSG 10 mg/kg plus ALN. After 12 weeks of treatment, bone quality was evaluated by mechanical testing. Microarchitecture, bone mineral density (BMD), cortical bone porosity, and bone remodeling were also measured.RESULTSOVX RSG 10 mg/kg rats had lower vertebral BMD and compromised trabecular architecture versus OVX controls. Increased cortical bone porosity and decreased mechanical properties occurred in these rats. ALN treatment prevented decreased BMD and architectural and mechanical properties in the OVX model. Reduced bone formation, increased marrow adiposity, and excess bone resorption were observed in RSG-treated rats.CONCLUSIONSRSG decreases bone quality. An unusual finding was an increase in cortical bone porosity induced by RSG, consistent with its effect on long bones of women. ALN, an inhibitor of bone resorption, enhanced mechanical strength and may provide an approach to partially counter the deleterious skeletal effects of RSG.

AMG 837: A Novel GPR40/FFA1 Agonist that Enhances Insulin Secretion and Lowers Glucose Levels in Rodents

Agonists of GPR40 (FFA1) have been proposed as a means to treat type 2 diabetes. Through lead optimization of a high throughput screening hit, we have identified a novel GPR40 agonist called AMG 837. The objective of these studies was to understand the preclinical pharmacological properties of AMG 837. The activity of AMG 837 on GPR40 was characterized through GTPγS binding, inositol phosphate accumulation and Ca2+ flux assays. Activity of AMG 837 on insulin release was assessed on isolated primary mouse islets. To determine the anti-diabetic activity of AMG 837 in vivo, we tested AMG 837 using a glucose tolerance test in normal Sprague-Dawley rats and obese Zucker fatty rats. AMG 837 was a potent partial agonist in the calcium flux assay on the GPR40 receptor and potentiated glucose stimulated insulin secretion in vitro and in vivo. Acute administration of AMG 837 lowered glucose excursions and increased glucose stimulated insulin secretion during glucose tolerance tests in both normal and Zucker fatty rats. The improvement in glucose excursions persisted following daily dosing of AMG 837 for 21-days in Zucker fatty rats. Preclinical studies demonstrated that AMG 837 was a potent GPR40 partial agonist which lowered post-prandial glucose levels. These studies support the potential utility of AMG 837 for the treatment of type 2 diabetes.

Obesity augments the age‐induced increase in mitochondrial capacity for H2O2 release in Zucker fatty rats

Mitochondrial dysfunction has been suggested to play a significant role in obesity and insulin resistance. The aim of the present study was to investigate if changes in obesity and insulin resistance were related to similar changes in mitochondrial capacity for hydrogen peroxide release in Zucker diabetic fatty rats and their lean littermates. Thirty-four rats were used in this study. Rats were either lean or obese Zucker rats killed at 5-6 (young) or 12-14 (adults) weeks of age. Mitochondria were isolated from soleus muscles; respiration and release of hydrogen peroxide were determined and related to citrate synthase activity to determine intrinsic mitochondrial function. Mitochondrial-specific super-oxide dismuthase (MnSOD) protein content was determined in isolated mitochondria and muscle homogenate. Catalase protein content was determined in muscle homogenate. Young lean and obese rats had a higher mitochondrial respiration when using palmitoyl-l-carnitine as substrate compared with adult lean and obese rats. The obese strain had higher mitochondrial hydrogen peroxide release but only in the adult animals. In both lean and obese animals, increased age was associated with increased mitochondrial hydrogen peroxide release. MnSOD tended to be higher in the obese strain in the isolated mitochondria. Regardless of age, catalase protein content was significantly lower in the obese rats. This study shows that the augmented increase in obesity and insulin resistance seen in Zucker diabetic fatty rats is associated with increased capacity for mitochondrial hydrogen peroxide release.

Blueberry Intake Alters Skeletal Muscle and Adipose Tissue Peroxisome Proliferator-Activated Receptor Activity and Reduces Insulin Resistance in Obese Rats

Metabolic syndrome can precede the development of type 2 diabetes and cardiovascular disease and includes phenotypes such as obesity, systemic inflammation, insulin resistance, and hyperlipidemia. A recent epidemiological study indicated that blueberry intake reduced cardiovascular mortality in humans, but the possible genetic mechanisms of this effect are unknown. Blueberries are a rich source of anthocyanins, and anthocyanins can alter the activity of peroxisome proliferator-activated receptors (PPARs), which affect energy substrate metabolism. The effect of blueberry intake was assessed in obesity-prone rats. Zucker Fatty and Zucker Lean rats were fed a higher-fat diet (45% of kcal) or a lower-fat diet (10% of kcal) containing 2% (wt/wt) freeze-dried whole highbush blueberry powder or added sugars to match macronutrient and calorie content. In Zucker Fatty rats fed a high-fat diet, the addition of blueberry reduced triglycerides, fasting insulin, homeostasis model index of insulin resistance, and glucose area under the curve. Blueberry intake also reduced abdominal fat mass, increased adipose and skeletal muscle PPAR activity, and affected PPAR transcripts involved in fat oxidation and glucose uptake/oxidation. In Zucker Fatty rats fed a low-fat diet, the addition of blueberry also significantly reduced liver weight, body weight, and total fat mass. Finally, Zucker Lean rats fed blueberry had higher body weight and reduced triglycerides, but all other measures were unaffected. In conclusion, whole blueberry intake reduced phenotypes of metabolic syndrome in obesity-prone rats and affected PPAR gene transcripts in adipose and muscle tissue involved in fat and glucose metabolism.

TS-071 is a novel, potent and selective renal sodium-glucose cotransporter 2 (SGLT2) inhibitor with anti-hyperglycaemic activity

The renal sodium-glucose cotransporter 2 (SGLT2) plays an important role in the reuptake of filtered glucose in the proximal tubule and therefore may be an attractive target for the treatment of diabetes mellitus. This study characterizes the pharmacological profile of TS-071 ((1S)-1,5-anhydro-1-[5-(4-ethoxybenzyl)-2-methoxy-4-methylphenyl]-1-thio-D-glucitol hydrate), a novel SGLT2 inhibitor in vitro and in vivo. Inhibition of glucose uptake by TS-071 was studied in CHO-K1 cells stably expressing either human SGLT1 or SGLT2. Single oral dosing studies were performed in rats, mice and dogs to assess the abilities of TS-071 to increase urinary glucose excretion and to lower plasma glucose levels. TS-071 inhibited SGLT2 activity in a concentration-dependent manner and was a potent and highly selective inhibitor of SGLT2. Orally administered TS-071 increased urinary glucose excretion in Zucker fatty rats and beagle dogs at doses of 0.3 and 0.03 mg·kg(-1) respectively. TS-071 improved glucose tolerance in Zucker fatty rats without stimulating insulin secretion and reduced hyperglycaemia in streptozotocin (STZ)-induced diabetic rats and db/db mice at a dose of 0.3 mg·kg(-1). These data indicate that TS-071 is a potent and selective SGLT2 inhibitor that improves glucose levels in rodent models of type 1 and 2 diabetes and may be useful for the treatment for diabetes mellitus.

2-({6-[(3R)-3-amino-3-methylpiperidine-1-yl]-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrrolo[3,2-d]pyrimidine-5-yl}methyl)-4-fluorobenzonitrile (DSR-12727): A potent, orally active dipeptidyl peptidase IV inhibitor without mechanism-based inactivation of CYP3A

We report on the identification of 2-({6-[(3R)-3-amino-3-methylpiperidine-1-yl]-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrrolo[3,2-d]pyrimidine-5-yl}methyl)-4-fluorobenzonitrile (DSR-12727) (7a) as a potent and orally active DPP-4 inhibitor without mechanism-based inactivation of CYP3A. Compound 7a showed good DPP-4 inhibitory activity (IC50=1.1nM), excellent selectivity against related peptidases and other off-targets, good pharmacokinetic and pharmacodynamic profile, great in vivo efficacy in Zucker-fatty rat, and no safety concerns both in vitro and in vivo.

Protection by Red Wine Polyphenols against Metabolic and Cardiovascular Alterations Associated with Obesity: A Possible Link with Estrogen Alpha Receptor

Epidemiological studies report an inverse association between dietary flavonoid consumption and mortality from cardiovascular and metabolic diseases. Red wine contains a wide variety of polyphenols (RWPC), which mainly derive from grape solids (skin and seeds) and can be divided into two classes, flavonoids and non-flavonoids. RWPC exert numerous effects including antioxidant and free radical properties, anti-platelet aggregating and anti-thrombotic activities. Moreover, RWPC are powerful vasodilators and contribute to the preservation of the integrity of the endothelium and to the inhibition of smooth muscle cell proliferation and migration. All these effects of red wine may interfere with atherosclerotic plaque development and stability, vascular thrombosis and occlusion and explain the prevention of ischemic heart disease, stroke and metabolic diseases observed in different experimental models. With regard to obesity, we studied the effects of dietary supplementation of RWPC in Zucker fatty rats (ZF). We show that RWPC lead to an improvement of glucose and lipid metabolism in ZF rats, enhances cardiac function and decreases peripheral resistance. In addition, RWPC improve endothelium-dependent relaxation in both aorta and small mesenteric arteries from ZF rats, an effect accompanied by an increase of vascular nitric oxide (NO) production and a decrease of superoxide anions release, resulting in an increase of NO bioavailability. Thus, RWPC lead to an overall improvement in obesity-associated alterations, including glucose and lipid metabolism, as well as endothelial and cardiac functions. A molecular basis of the effect of RWPC has been provided on the endothelium, indentifying the alpha isoform of the estrogen receptor (ERα) as the key receptor, or at least one of those transducing vascular effects exerted by these compounds, particularly delphinidin with respect to NO production. These results are of importance inasmuch as RWPC, by interacting with ERα, can activate other targets such the sirtuin-1-adenosine monophosphate protein kinase network, both of which are amongst the most potent regulators of cellular metabolism, including vascular cells, adipocytes and hepatocytes. Altogether, the available evidence indicates that RWPC might be of therapeutic benefit for cardiovascular and metabolic protections, although prospective controlled clinical trials are still needed.

Insulin-Regulated Srebp-1c and Pck1 mRNA Expression in Primary Hepatocytes from Zucker Fatty but Not Lean Rats Is Affected by Feeding Conditions

Insulin regulates the transcription of genes for hepatic glucose and lipid metabolism. We hypothesized that this action may be impaired in hepatocytes from insulin resistant animals. Primary hepatocytes from insulin sensitive Zucker lean (ZL) and insulin resistant Zucker fatty (ZF) rats in ad libitum or after an overnight fasting were isolated, cultured and treated with insulin and other compounds for analysis of gene expression using real-time PCR. The mRNA levels of one insulin-induced (Srebp-1c) and one insulin-suppressed (Pck1) genes in response to insulin, glucagon, and compactin treatments in hepatocytes from ad libitum ZL and ZF rats were analyzed. Additionally, the effects of insulin and T1317 on their levels in hepatocytes from ad libitum or fasted ZL or ZF rats were compared. The mRNA levels of Srebp-1c, Fas, and Scd1, but not that of Insr, Gck and Pck1, were higher in freshly isolated hepatocytes from ad libitum ZF than that from ZL rats. These patterns of Srebp-1c and Pck1 mRNA levels remained in primary hepatocyte cultured in vitro. Insulin's ability to regulate Srebp-1c and Pck1 expression was diminished in hepatocytes from ad libitum ZF, but not ZL rats. Glucagon or compactin suppressed Srebp-1c mRNA expression in lean, but not fatty hepatocytes. However, glucagon induced Pck1 mRNA expression similarly in hepatocytes from ad libitum ZL and ZF rats. Insulin caused the same dose-dependent increase of Akt phosphorylation in hepatocytes from ad libitum ZL and ZF rats. It synergized with T1317 to induce Srebp-1c, and suppressed Pck1 mRNA levels in hepatocytes from fasted, but not that from ad libitum ZF rats. We demonstrated that insulin was unable to regulate its downstream genes' mRNA expression in hepatocytes from ad libitum ZF rats. This impairment can be partially restored in hepatocytes from ZF rats after an overnight fasting, a phenomenon that deserves further investigation.

Altered gap junctional communication and renal haemodynamics in Zucker fatty rat model of type 2 diabetes

We examined the link between altered gap junctional communication and renal haemodynamic abnormalities in diabetes in studies performed on Zucker lean (ZL) and the Zucker diabetic fatty (ZDF) rat model of type 2 diabetes. The abundance of connexin (Cx) 37, 40 and 43 was assessed by western blot and immunohistochemistry. Renal haemodynamics was characterised with GAP peptides, which are Cx mimetics, to inhibit gap junctions as a probe in both strains. ZDF rats exhibited higher plasma glucose, 8-epi-prostaglandin F2α excretion, renal plasma flow and GFR than ZL rats. In ZDF rat kidney phosphorylation of Cx43 was enhanced compared with that in ZL rats. Immunohistochemical study revealed that the density of abundance of Cx37 in renin-secreting cells was significantly reduced in ZDF rats. Although renal autoregulation was markedly impaired in ZDF rats, it was preserved in ZL rats. GAP27 for Cx37,43 and for Cx40 impaired renal autoregulation in ZL rats, but failed to induce further alterations in renal autoregulation in ZDF rats. Our findings indicate that ZDF rats have glomerular hyperfiltration with impaired autoregulation. They also demonstrate enhanced phosphorylation of Cxs and reduced production of Cxs in ZDF rat kidney, especially of Cx37 in renin-secreting cells. Finally, our data suggest that an impairment of gap junctional communication in juxtaglomerular apparatus plays a role in altered renal autoregulation in diabetes.

Amlodipine and atorvastatin exert protective and additive effects via antiapoptotic and antiautophagic mechanisms after transient middle cerebral artery occlusion in Zucker metabolic syndrome rats

We examined the neuroprotective effects amlodipine and/or atorvastatin in metabolic syndrome (MetS) Zucker fatty rats against transient (90 min) middle cerebral artery occlusion (MCAO). The rats were pretreated with vehicle, amlodipine, atorvastatin, or amlodipine plus atorvastatin for 28 days, and 24 hr after transient MCAO the infarct size was assessed via hematoxylin and eosin staining, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) and microtubule-associated protein 1 light chain 3 (LC3) expression were examined by immunohistochemistry to evaluate apoptosis and autophagy, respectively. Compared with the vehicle group, rats treated with amlodipine or atorvastatin alone showed a significant decrease in infarct volume (P < 0.01), which was further decreased in the amlodipine plus atorvastatin group (P < 0.001). Compared with the vehicle group, the numbers of TUNEL- and LC3-positive cells were markedly reduced by amlodipine or atorvastatin alone (P < 0.01) and further decreased by amlodipine plus atorvastatin (P < 0.001). The number of apoptotic TUNEL/autophagic LC3 double-positive cells was also significantly decreased with amlodipine or atorvastatin alone compared with vehicle (P < 0.01) and was further decreased by amlodipine plus atorvastatin (P < 0.001). These data suggest additive neuroprotective effects of combination amlodipine and atorvastatin treatment after acute ischemic stroke in MetS model Zucker rats. These effects are mediated, at least in part, via antiapoptotic and antiautophagic mechanisms. Further studies are now needed to expand these preliminary results to understand fully the mechanisms involved in the protective effects of amlodipine and atorvastatin against ischemic stroke.

Observation of Lipid and Bone Metabolism in the Fatty Zucker Rats after Black Rice Administration

Observation of Lipid and Bone Metabolism in the Fatty Zucker Rats after Black Rice Administration

Inhibition of microsomal triglyceride transfer protein improves insulin sensitivity and reduces atherogenic risk in Zucker fatty rats

1. Insulin-resistant states are commonly associated with a significantly higher risk of atherosclerosis. Insulin resistance has also been correlated with enhanced very low-density lipoprotein (VLDL) production, which is exacerbated by increased intestinal lipid synthesis and insulin-stimulated de novo lipogenesis. Microsomal triglyceride transfer protein (MTP) catalyses the critical step in the synthesis and secretion of VLDL and chylomicrons. The purpose of the present study was to test the hypothesis that chronic inhibition of MTP with a small molecule inhibitor would improve insulin sensitivity and reduce atherogenic risk in a genetic model of diabetic dyslipidaemia. 2. The in vivo activity of BMS-201038, a potent inhibitor of MTP, was evaluated in a model of hypertriglyceridemia induced by Triton WR1339 and corn oil in Zucker fatty rats. Triglyceride secretion rate was significantly reduced by a single dose of BMS-201038 by 35% at 0.3 mg/kg and 47% at 1 mg/kg, respectively. 3. Another group of Zucker fatty rats was dosed orally with BMS-201038 (0.3 and 1 mg/kg) for 14 days. Serum levels of triglycerides were reduced by 71% and 87%, non-esterified free fatty acids were reduced by 33% and 40%, and low-density lipoproteins by 26% and 29%, by 0.3 mg/kg and 1 mg/kg dose of BMS-201038, respectively. These serum lipid changes were accompanied by significant improvements in glucose tolerance and insulin sensitivity. In addition, lipid peroxidation in liver was reduced by 59% and 61%, and superoxide dismutase activity was increased by 11% and 45% by 0.3 mg/kg and 1 mg/kg dose of BMS-201038, respectively. Similar beneficial changes were found in aorta as well. 4. The present study provides evidence that inhibition of MTP with a small molecule inhibitor significantly improves dyslipidaemia associated with insulin resistance and reduces the atherosclerotic risk.

Spontaneously Diabetic Torii Leprfa(SDT Fatty) Rat: A Novel Model of Obese Type 2 Diabetes

Diabetic animal models play critical roles in the elucidation of the mechanisms of diabetes mellitus and the complications, and in the development of novel drugs as treatments. Spontaneously Diabetic Torii Lepr fa (SDT fatty) rat, established by introducing the fa allele of the Zucker fatty rat into SDT rat genome, is a new model of obese type 2 diabetes. We have investigated the characteristics of SDT fatty rats. Both male and female SDT fatty rats show overt obesity, and hyperglycemia and hyperlipidemia are observed at a young age as compared with SDT rats. With early incidence of diabetes mellitus, diabetic complications in SDT fatty rats were seen at younger ages than those in the SDT rats. Furthermore, we evaluated the pharmacological effects of anti-diabetic drugs, such as metformin, pioglitazone, and dipeptidyl peptidase-4 inhibitor on SDT fatty rats. SDT fatty rat is a useful model for analysis of metabolic disease and the evaluation of drugs related to metabolic disease.

Role of inflammation and insulin resistance in endothelial progenitor cell dysfunction.

OBJECTIVEEndothelial progenitor cells (EPCs) are decreased in number and function in type 2 diabetes. Mechanisms by which this dysfunction occurs are largely unknown. We tested the hypothesis that a chronic inflammatory environment leads to insulin signaling defects in EPCs and thereby reduces their survival. Modifying EPCs by a knockdown of nuclear factor-κB (NF-κB) can reverse the insulin signaling defects, improve EPC survival, and decrease neointimal hyperplasia in Zucker fatty rats postangioplasty.RESEARCH DESIGN AND METHODSEPCs from Zucker fatty insulin-resistant rats were cultured and exposed to tumor necrosis factor-α (TNF-α). Insulin signaling defects and apoptosis were measured in the presence and absence of an NF-κB inhibitor, BAY11. Then, EPCs were modified by a knockdown of NF-κB (RelA) and exposed to TNF-α. For in vivo experiments, Zucker fatty rats were given modified EPCs post–carotid angioplasty. Tracking of EPCs was done at various time points, and neointimal hyperplasia was measured 3 weeks later.RESULTSInsulin signaling as measured by the phosphorylated–to–total AKT ratio was reduced by 56% in EPCs exposed to TNF-α. Apoptosis was increased by 71%. These defects were reversed by pretreatment with an NF-κB inhibitor, BAY11. Modified EPCs exposed to TNF-α showed a lesser reduction (RelA 20%) in insulin-stimulated AKT phosphorylation versus a 55% reduction in unmodified EPCs. Apoptosis was 41% decreased for RelA knockdown EPCs. Noeintimal hyperplasia postangioplasty was significantly less in rats receiving modified EPCs than in controls (intima-to-media ratio 0.58 vs. 1.62).CONCLUSIONSIn conclusion, we have shown that insulin signaling and EPC survival is impaired in Zucker fatty insulin resistant rats. For the first time, we have shown that this defect can be significantly ameliorated by a knockdown of NF-κB and that these EPCs given to Zucker fatty rats decrease neointimal hyperplasia post–carotid angioplasty.

Changes in renal vessels following the long-term administration of an angiotensin II receptor blocker in Zucker fatty rats

The nephro-protective effects of angiotensin II receptor blockers (ARBs) are widely known; however, there are few reports of long-term effects focusing on the renal vessels. We studied afferent arteriolar changes induced by the long-term administration of an ARB. Thirty-two 6-week-old male Zucker fatty rats (ZFRs) were divided into following four groups (n = 8 in each): ZFR Group and ZFR+High Group fed a standard or high-salt diet, respectively; ZFR+ARB Group and ZFR+High+ARB Group fed a standard or high-salt diet with ARB (Olmesartan, 5 mg/kg/day), respectively. Blood pressure, proteinuria, morphological examinations and glomerular haemodynamics in vivo were studied. Marked proliferative changes in the afferent arteriolar smooth muscle cells (SMCs) were frequently observed in the two groups given ARBs; in the ZFR+ARB group (77.3±10.3%) compared with the two groups without ARB (1.7%, p < 0.005; 1.2%, p < 0.0005) and 37.4±15.6% in the ZFR+High+ARB group. Proteinuria markedly decreased in the groups treated with ARBs, but the glomerular erythrocyte velocities showed no differences. Our findings indicate that long-term ARB administration induced unusual proliferative changes in SMCs of afferent arterioles of ZFRs. These changes could narrow arteriolar lumens and reduce intraglomerular pressure, but they could cause also irreversible damage to the arterioles.

Cardiovascular disease in recent onset diabetes mellitus

Diabetes is associated with a marked increased risk of atherosclerotic vascular disorders, including coronary, cerebrovascular, and peripheral artery disease. Cardiovascular disease (CVD) could account for disabilities and high mortality rates in patients with diabetes. Conventional risk factors, including hyperlipidemia, hypertension, smoking, obesity, lack of exercise, and a positive family history, contribute similarly to macrovascular complications in type 2 diabetic patients and non-diabetic subjects. The levels of these factors in diabetic patients are certainly increased, but not enough to explain the exaggerated risk for macrovascular complications in the diabetic population. Furthermore, recently, macrovascular complications of diabetes have been shown to start before the onset of diabetes. Indeed, several clinical studies have confirmed the increased risk of CVD in patients with impaired glucose tolerance (IGT). Since insulin resistance-related postprandial metabolic derangements are thought to play a central role in the development and progression of CVD in patients with IGT, amelioration of postprandial metabolic disturbance is a therapeutic target for the prevention of CVD in these high-risk patients. Therefore, in this paper, we review the molecular mechanisms for the increased risk of CVD in recent onset diabetes mellitus, especially focusing on postprandial dysmetabolism. We also discuss here the potential therapeutic strategies that specially target the mechanisms responsible for vascular alterations in diabetes.

Soluble fiber-enriched diets improve inflammation and oxidative stress biomarkers in Zucker fatty rats

In this study we evaluated the effect of the administration of different soluble fiber enriched-diets on inflammatory and redox state of Zucker fatty rats. Four groups of ten 8 week-old female Zucker fatty rats were used. The four groups were respectively fed the following diets until the 15th week of life: standard diet (obese control), 10% high methoxylated apple pectin (HMAP)-, 5% soluble cocoa fiber (SCF)-, and 10% β-glucan-enriched diets. A group of Zucker lean rats fed the standard diet was also used as control for normal values of this rat strain. The plasma levels of tumoral necrosis factor-α (TNF-α), adiponectin, and malondialdehyde (MDA) were measured at the end of treatment. The reduced glutathione liver levels were also obtained at that moment. TNF-α plasma levels decreased somewhat in Zucker fatty rats fed the different fibers, and MDA plasma levels significantly decreased in these animals. Nevertheless, adiponectin plasma levels increased in the Zucker fatty rats fed the SCF enriched diet, but did not change in the HMAP and the β-glucan group. The Zucker fatty rats fed the different fiber showed a trend towards increased the reduced glutathione liver levels, but significant differences with obese control rats were only obtained in the β-glucan group. The results obtained in this study suggest that the intake of the different soluble fiber-enriched diets that we have evaluated could prevent and/or attenuate the inflammatory and/or the prooxidative state of the metabolic syndrome.

Altered alkaline phosphatase activity in obese Zucker rats liver respect to lean Zucker and Wistar rats discussed in terms of all putative roles ascribed to the enzyme

Biliary complications often lead to acute and chronic liver injury after orthotopic liver transplantation (OLT). Bile composition and secretion depend on the integrated action of all the components of the biliary tree, starting from hepatocytes. Fatty livers are often discarded as grafts for OLT, since they are extremely vulnerable to conventional cold storage (CS). However, the insufficiency of donors has stimulated research to improve the usage of such marginal organs as well as grafts. Our group has recently developed a machine perfusion system at subnormothermic temperature (20°C; MP20) that allows a marked improvement in preservation of fatty and even of normal rat livers as compared with CS. We sought to evaluate the response of the biliary tree of fatty liver to MP20, and a suitable marker was essential to this purpose. Alkaline phosphatase (AlkP, EC 3.1.3.1), frequently used as marker of membrane transport in hepatocytes and bile ducts, was our first choice. Since no histochemical data were available on AlkP distribution and activity in fatty liver, we have first settled to investigate AlkP activity in the steatotic liver of fatty Zucker rats (fa/fa), using as controls lean Zucker (fa/+) and normal Wistar rats. The AlkP reaction in Wistar rats was in accordance with the existing data and, in particular, was present in bile canaliculi of hepatocytes in the periportal region and midzone, in the canals of Hering and in small bile ducts but not in large bile ducts. In lean ZR liver the AlkP reaction in Hering canals and small bile ducts was similar to Wistar rat liver but hepatocytes had lower canalicular activity and besides presented moderate basolateral reaction. The difference between lean Zucker and Wistar rats, both phenotypically normal animals, could be related to the fact that lean Zucker rats are genotypically heterozygous for a recessive mutated allele. In fatty liver, the activity in ductules and small bile ducts was unchanged, but most hepatocytes were devoid of AlkP activity with the exception of clusters of macrosteatotic hepatocytes in the mid-zone, where the reaction was intense in basolateral domains and in distorted canaliculi, a typical pattern of cholestasis. The interpretation of these data was hindered by the fact that the physiological role of AlkP is still under debate. In the present study, the various functions proposed for the role of the enzyme in bile canaliculi and in cholangiocytes are reviewed. Independently of the AlkP role, our data suggest that AlkP does not seem to be a reliable marker to study the initial step of bile production during OLT of fatty livers, but may still be used to investigate the behaviour of bile ductules and small bile ducts.